ABSTRACT

Introduction

Hereditary spherocytosis (HS) is a congenital hemolytic anemia caused by erythrocyte membrane protein defects. While the eosin‐5′‐maleimide (EMA) binding test by flow cytometry is the established first‐line assay, this study evaluates its optimal cut‐offs and performance within a large, real‐world cohort.

Methods

We retrospectively analyzed 181 consecutive samples (2017–2024) with suspected HS. EMA binding was performed by flow cytometry using conventional and standardized protocols; the latter (implemented in 2023) utilized fixed erythrocyte counts and FL1 bead calibration. Accuracy was assessed against a composite clinical–laboratory reference standard. ROC analysis determined sensitivity, specificity, likelihood ratios, and AUC.

Results

HS was confirmed in 50/181 cases (27.6%). EMA fluorescence reduction was significantly greater in HS (median 23.0%) than in non‐HS samples (9.0%). The optimal cut‐off (16.5%) yielded 96.0% sensitivity, 96.9% specificity, and AUC = 0.990 (95% CI 0.979–0.998). Both conventional and standardized protocols showed equivalent accuracy (AUC 0.988 vs. 0.992). False positives were mainly associated with severe microcytosis or autoimmune hemolytic anemia.

Conclusions

In a consecutive, real‐world cohort of patients with suspected hereditary spherocytosis referred to a single reference laboratory receiving samples from across the country, the EMA test demonstrated high diagnostic accuracy consistent with previous reports. Importantly, implementation of a standardized protocol resolved reproducibility issues in fluorescence intensity measurement without compromising diagnostic performance, supporting its robustness and applicability in routine clinical laboratories.